The present invention relates to a method for disinfecting immobilized enzymes, such as immobilized enzymes used for hydrolyzing lactose to glucose and galactose.
In general, the present invention can be used with any immobilized enzyme as long as the immobilized enzyme is not adversely affected by the disinfecting agent. For simplicity, however, the discussion which follows is limited to immobilized enzymes employed in the hydrolytic conversion of lactose to glucose and galactose, but is not to be construed as in any way limiting the spirit and scope of the invention.
In 1976, French cheese production reached approximately one million tons, resulting in the production of more than six million tons of whey as a by-product. Such whey contains, per liter, approximately 6 to 9 g. of protein, 45 to 50 g. of lactose, 6 to 8 g. of mineral salts, and 1 to 2 g. of fat. World-wide, the amount of lactose available from whey alone in 1977 totaled almost 3.5 million tons.
In the past, whey was regarded as a waste product and was discharged into sewers or streams and rivers. Because of increasing concerns over environmental pollution, much of the whey is being processed into a variety of animal and human foods. For example, the development of high performance ultrafilters now permits the separation of whey protein from the whey. Such protein is of exceptional value. For example, a 35 percent concentrate of whey protein can replace nonfat dry milk in many food products, such as baked goods, beverages, and frozen desserts. Unfortunately, the separation of proteins from whey results in a liquid fraction, called permeate, which until recently has had little or no value.
With the advent of immobilized enzymes, however, hydrolysis of the lactose in the permeate, either with or without demineralization, has become commercially feasible. Because of the presence of glucose and galactose, hydrolyzed lactose is much sweeter and more soluble than lactose alone. Thus, the hydrolyzed product is a functional sweetener which can be used in the preparation of pastries, milk-based desserts, and frozen confections such as ice cream. Furthermore, the hydrolyzed product is an efficiently fermentable mixture suitable for use as a fermentation substrate in, for example, the brewing and pharmaceutical industries.
It also is possible to carry out the hydrolysis of the lactose in whey without a prior ultrafiltration step and either with or without demineralization. The product, which still contains proteins, is similar to hydrolyzed permeate and, consequently, it also can be used in the food industry as already described.
Various methods for hydrolyzing lactose are, of course, well known to those having ordinary skill in the art. Enzymatic hydrolysis is especially useful for the production of food-related products and, as already indicated, the use of immobilized enzymes is particularly attractive.
By way of illustration only, H. H. Weetall et al., Biotechnol. Bioeng., 16, 295 (1974), discuss the preparation of immobilized lactase and its use in the enzymatic hydrolysis of acid whey. The enzyme, isolated from both fungi and yeast, was immobilized on zirconia-coated porous glass particles. The substrate consisted of either aqueous lactose solution or acid whey permeate.
Additionally, L. E. Wierzbicki et al., Biotechnol. Bioeng. 16, 397 (1974), reported on the hydrolysis of lactose in acid whey using lactase (.beta.-galactosidase) immobilized on porous glass particles with emphasis on the preparation and characterization of a reusable catalyst for the production of low-lactose dairy products. Partially purified lactases from Aspergillus niger, Lactobacillus helveticus, and Saccharomyces lactis were immobilized on porous glass particles. The substrate consisted of acid whey powder which had been reconstituted in water to the appropriate solids concentration. In some instances, the reconstituted acid whey was deproteinized by heating in a boiling water bath for five minutes.
Finally, H. H. Weetall et al., Biotechnol. Bioeng., 16, 689 (1974), describe the preparation of immobilized lactase as part of continued studies on the enzymatic hydrolysis of lactose. A fungal lactase was employed, immobilized on zirconia-coated controlled-pore glass and porous titania particles. The resulting immobilized enzyme preparations were used for the hydrolysis of lactose in whole sweet whey, whole acid whey, acid whey ultrafiltrate, and pure lactose.
As noted by H. H. Weetall et al., Biotechnol. Bioeng., 16, 689 (1974), bacterial contamination of the whey substrate was a major problem. The continued growth of microorganisms clogged the columns. The use of 2 percent gluteraldehyde at pH 4.0 was found to kill the bacteria without destroying the activity of the immobilized enzyme. These authors stated that a sanitizing procedure would be absolutely necessary in any plant situation.
It is apparent, however, that any commercially-viable sanitizing or disinfecting procedure must effectively destroy substantially all of the contaminating microorganisms without any appreciable harmful effect on the immobilized enzymes. Furthermore, in cases where the product is intended for use in the food industry, the disinfecting agent often must meet governmental regulatory requirements.
Until now, no truly satisfactory disinfectant has been available. Acetic acid, which is commonly used as a dilute aqueous solution, gives only fair results. Moreover, other known disinfectants, such as halogen derivatives, organic acids, quaternary ammonium compounds, biguanidine polymers, and the like tend to be unsuitable because of partial or complete inactivation of the enzymes.
Thus, there still is a need for a commercially-viable sanitizing or disinfecting procedure for immobilized enzymes which is effective without significant deleterious effects on the immobilized enzymes.